Methods for modulating the axonal growth of central nervous system neurons

ABSTRACT

Methods for modulating the axonal outgrowth of central nervous system neurons are provided. Methods for stimulating the axonal outgrowth of central nervous system neurons following an injury (e.g., stroke, Traumatic Brain Injury, cerebral aneurism, spinal cord injury and the like) and methods for inhibiting the axonal outgrowth of central nervous system neurons are also provided. Finally, a packed formulation comprising a pharmaceutical composition comprising an inosine nucleoside and a pharmaceutically acceptable carrier packed with instructions for use of the pharmaceutical composition for treatment of a central nervous system disorder is provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. Ser. No. 11/132,701, filedMay 19, 2005,now pending, which is a Continuation of U.S. Ser. No.10/385,031, filed Mar. 10, 2003, now abandoned, which is a Continuationof U.S. Ser. No. 09/997,687 filed on Nov. 11, 2001, now abandoned, whichis a Continuation of U.S. Ser. No. 08/921,902 filed on Sep. 2, 1997,issued as U.S. Pat. No. 6,440,455.

GOVERNMENT FUNDING

Work described herein was supported, at least in part, under grantR01EY05690 awarded by the National Eye Institute. The U.S. governmenttherefore may have certain rights in this invention.

BACKGROUND OF THE INVENTION

Past early childhood, injury to the central nervous system (CNS) resultsin functional impairments that are largely irreversible. Within thebrain or spinal cord, damage resulting from stroke, trauma, or othercauses can result in life-long losses in cognitive, sensory and motorfunctions, and even maintenance of vital functions. Nerve cells that arelost are not replaced, and those that are spared are generally unable toregrow severed connections, although a limited amount of local synapticreorganization can occur close to the site of injury. Functions that arelost are currently untreatable.

Regenerative failure in the CNS has been attributed to a number offactors, which include the presence of inhibitory molecules on thesurface of glial cells that suppress axonal growth; absence ofappropriate substrate molecules such as laminin to foster growth and anabsence of the appropriate trophic factors needed to activate programsof gene expression required for cell survival and differentiation.

By contrast, within the peripheral nervous system (PNS), injured nervefibers can regrow over long distances, with eventual excellent recoveryof function. Within the past 15 years, neuroscientists have come torealize that this is not a consequence of intrinsic differences betweenthe nerve cells of the peripheral and central nervous system;remarkably, neurons of the CNS will extend their axons over greatdistances if given the opportunity to grow through a grafted segment ofPNS (e.g., sciatic nerve). Therefore, neurons of the CNS retain acapacity to grow if given the right signals from the extracellularenvironment. Factors which contribute to the differing growth potentialsof the CNS and PNS include partially characterized, growth-inhibitingmolecules on the surface of the oligodendrocytes that surround nervefibers in the CNS, but which are less abundant in the comparable cellpopulation of the PNS (Schwann cells); molecules of the basal lamina andother surfaces that foster growth in the PNS but which are absent in theCNS (e.g., laminin); and trophic factors, soluble polypeptides whichactivate programs of gene expression that underlie cell survival anddifferentiation. Although such trophic factors are regarded as essentialfor maintaining the viability and differentiation of nerve cells, theparticular ones that are responsible for inducing axonal regeneration inthe CNS remain uncertain. As a result, to date, effective treatments forCNS injuries have not been developed.

Accordingly, methods and compositions for modulating the outgrowth ofCNS neurons are still needed.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for modulatingthe axonal outgrowth of central nervous system neurons. The invention isbased, at least in part, on the discovery that purine nucleosides andanalogs thereof are capable of modulating (i.e. either stimulating orinhibiting) axonal outgrowth of CNS neurons. Accordingly, the method ofthe invention involves contacting central nervous system neurons with apurine nucleoside or analog thereof. In one aspect, the inventionprovides methods for stimulating outgrowth, preferably using inosine orguanosine nucleosides or analogs thereof. In another aspect, theinvention provides methods for inhibiting outgrowth, preferably using a6-thioguanine nucleoside. In a preferred embodiment, the methods of theinvention modulate axonal outgrowth of retinal ganglion cells.

The invention also provides methods for stimulating the outgrowth ofcentral nervous system neurons following damage or other injury to theCNS neurons (e.g., stroke, Traumatic Brain Injury, cerebral aneurism,spinal cord injury and the like). These methods involve administering toa subject a purine nucleoside (e.g., inosine or guanosine), or analogthereof, such that axonal outgrowth is stimulated. In one aspect, thepurine nucleoside or analog thereof is administered by introduction intothe central nervous system of the subject, for example into thecerebrospinal fluid of the subject. In certain aspects of the invention,the purine nucleoside or analog thereof is introduced intrathecally, forexample into a cerebral ventricle, the lumbar area, or the cisternamagna. In a preferred embodiment, the stimulatory method of theinvention promotes outgrowth of damaged retinal ganglion cells. Thepurine nucleoside or analog thereof can be administered locally toretinal ganglion cells to stimulate axonal outgrowth.

In another embodiment, the invention provides methods for inhibitingoutgrowth of CNS neurons in which a purine nucleoside (e.g.,6-thioguanine) is administered to a subject. The inhibitory methods ofthe invention can be used to inhibit axonal outgrowth in, for example,neuroproliferative disorders or neuropathic pain syndromes.

In yet another aspect of the invention, the purine nucleoside or analogthereof is administered in a pharmaceutically acceptable formulation.The pharmaceutically acceptable formulation can be a dispersion system,for example a lipid-based formulation, a liposome formulation, or amultivesicular liposome formulation. The pharmaceutically acceptableformulation can also comprise a polymeric matrix, selected, for example,from synthetic polymers such as polyesters (PLA, PLGA), polyethyleneglycol, poloxomers, polyanhydrides, and pluronics or selected fromnaturally derived polymers, such as albumin, alginate, cellulosederivatives, collagen, fibrin, gelatin, and polysaccharides.

In a further aspect of the invention, the pharmaceutically acceptableformulation provides sustained delivery, e.g., “slow release” of thepurine nucleoside to a subject for at least one, two, three, or fourweeks after the pharmaceutically acceptable formulation is administeredto the subject. Sustained delivery of a formulation of the invention maybe provided by use of, for example, slow release capsules or an infusionpump.

The invention, finally, provides a pharmaceutical composition comprisinga purine nucleoside or analog thereof and a pharmaceutically acceptablecarrier.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D are graphs showing the quantitation of purinergic effects onaxonal outgrowth.

FIG. 1A is a graph depicting axonal growth in response to thenucleosides adenosine (A), guanosine (G), cytidine (C), uridine (U), andthymidine (T) at a concentration of 1, 10, and 100 μM as indicated. Dataare normalized by subtracting the level of growth in the negativecontrols and then dividing by the net growth in positive controlstreated with 20-30% AF-1.

FIG. 1B is a graph depicting dose-response curves for adenosine andguanosine. EC₅₀ values estimated from these data are 10-15 μM foradenosine and 20-30 μM for guanosine.

FIG. 1C is a graph depicting the effects of adenosine nucleotides.

FIG. 1D is a graph depicting the effects of membrane-permeable analogsof cyclic AMP (dBCAMP, dibutyryl cyclic AMP; Sp-8-Br-cAMPS,8-bromoadenosine-3′,5′ cyclic monophosphorothioate) or cyclic GMP (8-BrcGMP, 8-bromo cyclic GMP; 8-pcpt-cGMP, 8-(4-chlorophenylthio)guanosine-3′,5′-cyclic monophosphate). Data represent means+standarderrors of the mean (SEM; not shown if <0.02) and are pooled from 2-4independent experiments. p values are based upon 2-tailed t-tests,comparing growth to that of the negative controls. *p<0.05; **p<0.01;***p<0.001.

FIG. 2 is a graph showing that adenosine does not stimulate growth viaextracellular receptors. Outgrowth stimulated by AF-1 (a-b), 100 μMadenosine (Ado) (c-d), or 100 μM guanosine (Guo) (e-f), is unaffected bythe addition of 20 pM 8-PST, an inhibitor of A1 and A2 adenosinereceptors (compare growth in a, c, and e with b, d, and f). Thenonhydrolyzable adenosine analog, 2-chloroadenosine (2-CA, 100 μM)diminishes growth below baseline levels (g) (p<0.001 in 3 experiments).

FIG. 3 is a graph showing that adenosine must be hydrolyzed to stimulateoutgrowth. Top: A graph depicting the effects of deoxycoformycin (DCF)and exogenous adenosine deaminase (ADA) on outgrowth induced by AF-1(a-c), adenosine (d-f), and guanosine (g, h). Bottom: A graph depictingthe effects of deoxycoformycin (DCF) and exogenous adenosine deaminase(ADA) on survival induced by AF-1 (a-c), adenosine (d-f), and guanosine(g,h). Whereas augmenting adenosine hydrolysis with exogenous ADA leavesthe activity of adenosine unaltered (f), blocking endogenous ADAactivity with DCF causes adenosine to suppress growth (e, top) andsurvival (e, bottom). ***p<0.001.

FIG. 4 is a graph depicting a dose-response curve for inosine. Atconcentrations above 50 μM, inosine stimulates about 60% the maximallevel of growth achieved with AF-1. The EC₅₀ for inosine is estimated tobe 10-15 μM. Hypoxanthine was inactive, while 5′ IMP appears to haveless than 1/10 the activity of inosine. Outgrowth stimulated by allconcentrations of inosine 10 μM is significantly above background(p<0.001).

FIG. 5 is a graph depicting that inosine and guanosine stimulate growththrough an intracellular mechanism. At 20 μM. NBTI, an inhibitor orpurine transport, has no effect on the activity of AF-1, but blocks c.90% of the activity of inosine (50 μM) or guanosine (100 μM). ***differences in growth with and without drugs are significant at p<0.001.Data is pooled from 4 independent experiments.

FIG. 6A is a graph showing that AF-1 contains no apparent inosineactivity. On a G-10 Sephadex column, AF-1 elutes with a peak of 7minutes, with no activity detected at the time of peak inosine elution(i.e., 9-10 min).

FIG. 6B is a graph showing that the effects of inosine and guanosine areindependent of cell density. Data from multiple independent experiments,each indicated by a single point, were analyzed for the effect ofplating density on cell outgrowth. In all cases, the concentration ofinosine or guanosine was maintained at 100 μM. The regression lines werecalculated by least-squares-fit (Cricket Graph) and are shown below thesymbols.

FIGS. 7A-D are graphs showing that the effects of AF-1 are inhibited by6-thioguanine but restored by inosine.

FIG. 7A shows that at 10 μM, the purine analog 6-TG suppressed growthinduced by AF-1 below baseline (lane 2 vs. 1:p<0.001) and reduced thegrowth induced by 25 μM inosine (Ino-25) by about 50% (lane 4 vs. 3);Growth induced by higher concentrations of inosine or guanosine(Guo-100: lanes 8 vs. 7) were unaffected. Inosine at 100 μM restored allof the growth induced by AF-1 in the presence of 10 μM 6-TG (lane 10),which is significantly higher than the growth induced by 100 μM inosine,either alone or with 10 μM 6-TG (p<0.01).

FIG. 7B is a graph showing that the concentration of 6-TG used here hadno effect on cell survival.

FIG. 7C is a graph showing that AF-1 and inosine have partially additiveeffects. Outgrowth was assessed for AF-1 and inosine, each at 0, EC₅₀,or saturating concentrations. While the effects of half-maximalconcentrations of each were additive (lane 5), growth reached a plateaulevel in the presence of higher concentrations of each (lanes 6, 8, 9).

FIG. 7D shows further studies on the effects of 6-thioguanine. Outgrowthstimulated by AF-1 was completely blocked by 6-TG (10 μM) and was notrestored in the presence of NBTI (N, 20 μM) and/or dipyridamole (D, 10μM), purine transport blockers inhibitors that suppress the activity ofinosine. Inhibitory effects of 6-TG were not mimicked by two reducingagents, a-tocopherol (a-toc, 30 μM) or glutathione a-methyl ester (MEG,100 μM).

FIG. 8 is a graph depicting the effects of purines on rat retinalganglion cells (quantitative studies). CNTF stimulated growth isinhibited by 6-TG (10 μM) but is fully restored by the addition of 25 μMinosine. Significance of differences from control: *p=0.03; ***p<0.001.Results are pooled from 3 independent studies.

DETAILED DESCRIPTION

The present invention provides methods for modulating the axonaloutgrowth of central nervous system neurons. The invention is based, atleast in part, on the discovery that purine nucleosides (e.g., inosineand guanosine) and analogs thereof induce stimulation of axonaloutgrowth from both goldfish as well as mammalian retinal ganglion cells(see Examples I and XI, respectively). As shown in Example II, purinenucleosides are more active than their nucleotide counterparts, and theyexert their effect through an intracellular pathway (see Example VI).The invention further is based, at least in part, on the discovery thatadenosine nucleosides and analogs thereof induce inhibition of axonaloutgrowth from retinal ganglion cells (see Example X).

Accordingly, the methods of the invention for modulating axonaloutgrowth of CNS neurons generally involve contacting the centralnervous system neurons with a purine nucleoside or analog thereof suchthat axonal outgrowth is modulated.

As used herein, the language “modulating the axonal outgrowth of centralnervous system neurons” is intended to include the capacity to stimulateor inhibit axonal outgrowth of central nervous system neurons to variouslevels, e.g., to levels which allow for the treatment of targeted CNSinjuries.

As used herein, the term “outgrowth” refers to the process by whichaxons grow out of a CNS neuron. The outgrowth can result in a totallynew axon or the repair of a partially damaged axon. Outgrowth istypically evidenced by extension of an axonal process of at least 5 celldiameters in length.

As used herein, the term “CNS neurons” is intended to include theneurons of the brain and the spinal cord which are unresponsive to nervegrowth factor (NGF). The term is not intended to include support orprotection cells such as astrocytes, oligodentrocytes, microglia,ependyma and the like, nor is it intended to include peripheral nervoussystem (e.g., somatic, autonomic, sympathetic or parasympathetic nervoussystem) neurons. Preferred CNS neurons are mammalian neurons, morepreferably human neurons.

As used herein, the language “contacting” is intended to include both invivo or in vitro methods of bringing a purine nucleoside or analogthereof into proximity with a CNS neuron, such that the purinenucleoside or analog thereof can modulate the outgrowth of axonalprocesses from said CNS neuron.

As used herein, the language “purine nucleoside” is art recognized andis intended to include any purine base linked to a sugar, or an analogthereof. For example, urine nucleosides include guanine, inosine oradenine and analogs include 6-thioguanine (6-TG) and the like.

In one embodiment, the outgrowth of CNS neurons is stimulated,preferably using inosine or guanosine nucleosides or analogs thereof. Inanother embodiment, the outgrowth of CNS neurons is inhibited,preferably using a 6-TG nucleoside.

The invention also provides methods for stimulating the outgrowth ofcentral nervous system neurons following an injury. The method involvesadministering to a subject a purine nucleoside (e.g., inosine orguanosine) or analog thereof.

As used herein, the term “subject” is intended to include animalssusceptible to CNS injuries, preferably mammals, most preferably humans.In a preferred embodiment, the subject is a primate. In an even morepreferred embodiment, the primate is a human. Other examples of subjectsinclude dogs, cats, goats, and cows.

As used herein, the term “injury” is intended to include a damage whichdirectly or indirectly affects the normal functioning of the CNS. Forexample, the injury can be damage to retinal ganglion cells; a traumaticbrain injury; a stroke related injury; a cerebral aneurism relatedinjury; a spinal cord injury, including monoplegia, diplegia,paraplegia, hemiplegia and quadriplegia; a neuroproliferative disorderor neuropathic pain syndrome.

As used herein, the term “stroke” is art recognized and is intended toinclude sudden diminution or loss of consciousness, sensation, andvoluntary motion caused by rapture or obstruction (e.g. by a blood clot)of an artery of the brain.

As used herein, the term “Traumatic Brain Injury” is art recognized andis intended to include the condition in which, a traumatic blow to thehead causes damage to the brain, often without penetrating the skull.Usually, the initial trauma can result in expanding hematoma,subarachnoid hemorrhage, cerebral edema, raised intracranial pressure(ICP), and cerebral hypoxia, which can, in turn, lead to severesecondary events due to low cerebral blood flow (CBF).

Pharmaceutically Acceptable Formulations

In the method of the invention, the purine nucleoside or analog thereofcan be administered in a pharmaceutically acceptable formulation. Thepresent invention pertains to any pharmaceutically acceptableformulations, such as synthetic or natural polymers in the form ofmacromolecular complexes, nanocapsules, microspheres, or beads, andlipid-based formulations including oil-in-water emulsions, micelles,mixed micelles, synthetic membrane vesicles, and resealed erythrocytes.

In one embodiment, the pharmaceutically acceptable formulations comprisea polymeric matrix.

The terms “polymer” or “polymeric” are art-recognized and include astructural framework comprised of repeating monomer units which iscapable of delivering a purine nucleoside or analog thereof such thattreatment of a targeted condition, e.g., a CNS injury, occurs. The termsalso include co-polymers and homopolymers e.g., synthetic or naturallyoccurring. Linear polymers, branched polymers, and cross-linked polymersare also meant to be included.

For example, polymeric materials suitable for forming thepharmaceutically acceptable formulation employed in the presentinvention, include naturally derived polymers such as albumin, alginate,cellulose derivatives, collagen, fibrin, gelatin, and polysaccharides,as well as synthetic polymers such as polyesters (PLA, PLGA),polyethylene glycol, poloxomers, polyanhydrides, and pluronics. Thesepolymers are biocompatible with the nervous system, including thecentral nervous system, they are biodegradable within the centralnervous system without producing any toxic byproducts of degradation,and they possess the ability to modify the manner and duration of purinenucleoside release by manipulating the polymer's kineticcharacteristics. As used herein, the term “biodegradable” means that thepolymer will degrade over time by the action of enzymes, by hydrolyticaction and/or by other similar mechanisms in the body of the subject. Asused herein, the term “biocompatible” means that the polymer iscompatible with a living tissue or a living organism by not being toxicor injurious and by not causing an immunological rejection.

Polymers can be prepared using methods known in the art (Sandier, S. R.;Karo, W. Polymer Syntheses; Harcourt Brace: Boston, 1994; Shalaby, W.;Ikada, Y.; Langer. R.; Williams, J. Polymers of Biological andBiomedical Significance (ACS Symposium Series 540; American ChemicalSociety: Washington, D.C., 1994). Polymers can be designed to beflexible; the distance between the bioactive side-chains and the lengthof a linker between the polymer backbone and the group can becontrolled. Other suitable polymers and methods for their preparationare described in U.S. Pat. Nos. 5,455,044 and 5,576,018, the contents ofwhich are incorporated herein by reference.

The polymeric formulations are preferably formed by dispersion of thepurine nucleoside within liquefied polymer, as described in U.S. Pat.No. 4,883,666, the teachings of which are incorporated herein byreference or by such methods as bulk polymerization, interfacialpolymerization, solution polymerization and ring polymerization asdescribed in Odian G., Principles of Polymerization and ring openingpolymerization, 2nd ed., John Wiley & Sons, New York, 1981, the contentsof which are incorporated herein by reference. The properties andcharacteristics of the formulations are controlled by varying suchparameters as the reaction temperature, concentrations of polymer andpurine nucleoside, types of solvent used, and reaction times.

In addition to the purine nucleoside and the pharmaceutically acceptablepolymer, the pharmaceutically acceptable formulation used in the methodof the invention can comprise additional pharmaceutically acceptablecarriers and/or excipients. As used herein, “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, coatings,antibacterial and anti fungal agents, isotonic and absorption delayingagents, and the like that are physiologically compatible. For example,the carrier can be suitable for injection into the cerebrospinal fluid.Excipients include pharmaceutically acceptable stabilizers anddisintegrants.

The purine nucleoside or analog thereof can be encapsulated in one ormore pharmaceutically acceptable polymers, to form a microcapsule,microsphere, or microparticle, terms used herein interchangeably.Microcapsules, microspheres, and microparticles are conventionallyfree-flowing powders consisting of spherical particles of 2 millimetersOr less in diameter, usually 500 microns or less in diameter. Particlesless than 1 micron are conventionally referred to as nanocapsules,nanoparticles or nanospheres. For the most part, the difference betweena microcapsule and a nanocapsule, a microsphere and a nanosphere, ormicroparticle and nanoparticle is size; generally there is little, ifany, difference between the internal structure of the two. In one aspectof the present invention, the mean average diameter is less than about45 μm, preferably less than 20 μm, and more preferably between about 0.1and 10 μm.

In another embodiment, the pharmaceutically acceptable formulationscomprise lipid-based formulations. Any of the known lipid-based drugdelivery systems can be used in the practice of the invention. Forinstance, multivesicular liposomes (MVL), multilamellar liposomes (alsoknown as multilamellar vesicles or “MLV”), unilamellar liposomes,including small unilamellar liposomes (also known as unilamellarvesicles or “SUV”) and large unilamellar liposomes (also known as largeunilamellar vesicles or “LUV”), can all be used so long as a sustainedrelease rate of the encapsulated purine nucleoside or analogue thereofcan be established. In one embodiment, the lipid-based formulation canbe a multivesicular liposome system. Methods of making controlledrelease multivesicular liposome drug delivery systems is described inPCT Application Serial Nos. US96/11642, US94/12957 and US94/04490, thecontents of which are incorporated herein by reference.

The composition of the synthetic membrane vesicle is usually acombination of phospholipids, usually in combination with steroids,especially cholesterol. Other phospholipids or other lipids may also beused.

Examples of lipids useful in synthetic membrane vesicle productioninclude phosphatidylglycerols, phosphatidylcholines,phosphatidylserines, phosphatidylethanolamines, sphingolipids,cerebrosides, and gangliosides. Preferably phospholipids including eggphosphatidylcholine, dipalmitoylphosphatidylcholine,distearoylphosphatidylcholine, dioleoylphosphatidylcholine,dipalmitoylphosphatidylglycerol, and dioleoylphosphatidylglycerol areused.

In preparing lipid-based vesicles containing a purine nucleoside oranalogue thereof, such variables as the efficiency of purine nucleosideencapsulation, lability of the purine nucleoside, homogeneity and sizeof the resulting population of vesicles, purine nucleoside-to-lipidratio, permeability, instability of the preparation, and pharmaceuticalacceptability of the formulation should be considered (see Szoka, etal., Annual Reviews of Biophysics and Bioengineering, 9:467, 1980;Deamer, et al., in Liposomes, Marcel Dekker, New York, 1983, 27, andHope, et al., Chem. Phys. Lipids, 40:89, 1986, the contents of which areincorporated herein by reference).

Administration of the Pharmaceutically Acceptable Formulation

In one embodiment, the purine nucleoside or analog thereof isadministered by introduction into the central nervous system of thesubject, e.g., into the cerebrospinal fluid of the subject. In certainaspects of the invention, the purine nucleoside or analog thereof isintroduced intrathecally, e.g., into a cerebral ventricle, the lumbararea, or the cisterna magna. In another aspect, the purine nucleoside oranalog thereof is introduced intraocullarly, to thereby contact retinalganglion cells.

The pharmaceutically acceptable formulations can easily be suspended inaqueous vehicles and introduced through conventional hypodermic needlesor using infusion pumps. Prior to introduction, the formulations can besterilized with, preferably, gamma radiation or electron beamsterilization, described in U.S. Pat. No. 436,742 the contents of whichare incorporated herein by reference.

In one embodiment, the purine nucleoside formulation described herein isadministered to the subject in the period from the time of injury to 100hours, for example within 24, 12 or 6 hours after the injury hasoccurred.

In another embodiment of the invention, the purine nucleosideformulation is administered into a subject intrathecally. As usedherein, the term “intrathecal administration” is intended to includedelivering a purine nucleoside formulation directly into thecerebrospinal fluid of a subject, by techniques including lateralcerebroventricular injection through a burrhole or cisternal or lumbarpuncture or the like (described in Lazorthes et al. Advances in DrugDelivery Systems and Applications in Neurosurgery, 143-192 and Omaya etal., Cancer Drug Delivery, 1: 169-179, the contents of which areincorporated herein by reference). The term “lumbar region” is intendedto include the area between the third and fourth lumbar (lower back)vertebrae. The term “cisterna magna” is intended to include the areawhere the skull ends and the spinal cord begins at the back of the head.The term “cerebral ventricle” is intended to include the cavities in thebrain that are continuous with the central canal of the spinal cord.Administration of a purine nucleoside to any of the above mentionedsites can be achieved by direct injection of the purine nucleosideformulation or by the use of infusion pumps. For injection, the purinenucleoside formulation of the invention can be formulated in liquidsolutions, preferably in physiologically compatible buffers such asHank's solution or Ringer's solution. In addition, the purine nucleosideformulation may be formulated in solid form and re-dissolved orsuspended immediately prior to use. Lyophilized forms are also included.The injection can be, for example, in the form of a bolus injection orcontinuous infusion (e.g., using infusion pumps) of the purinenucleoside formulation.

In one embodiment of the invention, said purine nucleoside formulationis administered by lateral cerebro ventricular injection into the brainof a subject in the inclusive period from the lime of the injury to 100hours thereafter. The injection can be made, for example, through a burrhole made in the subject's skull. In another embodiment, saidencapsulated therapeutic agent is administered through a surgicallyinserted shunt into the cerebral ventricle of a subject in the inclusiveperiod from the time of the injury to 100 hours thereafter. For example,the injection can be made into the lateral ventricles, which are larger,even though injection into the third and fourth smaller ventricles canalso be made.

In yet another embodiment, said purine nucleoside formulation isadministered by injection into the cisterna magna, or lumbar area of asubject in the inclusive period from the time of the injury to 100 hoursthereafter.

Duration and Levels of Administration

In another embodiment of the method of the invention, thepharmaceutically acceptable formulation provides sustained delivery,e.g., “slow release” of the purine nucleoside to a subject for at leastone, two, three, or four weeks after the pharmaceutically acceptableformulation is administered to the subject.

As used herein, the term “sustained delivery” is intended to includecontinual delivery of a purine nucleoside or analogue thereof in vivoover a period of time following administration, preferably at leastseveral days, a week or several weeks. Sustained delivery of the purinenucleoside or analogue thereof can be demonstrated by, for example, thecontinued therapeutic effect of the purine nucleoside or analoguethereof over time (e.g., sustained delivery of the purine nucleoside oranalogue thereof can be demonstrated by continued outgrowth or bycontinued inhibition of outgrowth of CNS neurons over time).Alternatively, sustained delivery of the purine nucleoside or analoguethereof may be demonstrated by detecting the presence of the purinenucleoside or analogue thereof in vivo over time.

In one embodiment, the pharmaceutically acceptable formulation providessustained delivery of the purine nucleoside or analogue thereof to asubject for less than 30 days after the purine nucleoside or analoguethereof is administered to the subject. For example, thepharmaceutically acceptable formulation, e.g., “slow release”formulation, can provide sustained delivery of the purine nucleoside oranalogue thereof to a subject for one, two, three or four weeks afterthe purine nucleoside or analogue thereof is administered to thesubject. Alternatively, the pharmaceutically acceptable formulation mayprovide sustained delivery of the purine nucleoside or analogue thereofto a subject for more than 30 days after the purine nucleoside oranalogue thereof is administered to the subject.

The pharmaceutical formulation, used in the method of the invention,contains a therapeutically effective amount of the purine nucleoside oranalogue thereof. A “therapeutically effective amount” refers to anamount effective, at dosages and for periods of time necessary, toachieve the desired result. A therapeutically effective amount of thepurine nucleoside or analogue thereof may vary according to factors suchas the disease state, age, and weight of the subject, and the ability ofthe purine nucleoside or analogue thereof (alone or in combination withone or more other agents) to elicit a desired response in the subject.Dosage regimens may be adjusted to provide the optimum therapeuticresponse. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the purine nucleoside or analoguethereof are outweighed by the therapeutically beneficial effects. Anon-limiting range for a therapeutically effective concentration ofinosine is 5 μM to 1 mM. A non-limiting range for a therapeuticallyeffective concentration of guanosine is at least 25 μM to 1 mM. In aparticularly preferred embodiment, the therapeutically effectiveconcentration of the inosine nucleoside is 10-25 μM, or 25-50 μM. In aparticularly preferred embodiment, the therapeutically effectiveconcentration of the guanosine nucleoside is 25-50 μM, 50-100 μM, or100-150 μM. Adenosine can be used to inhibit neurite outgrowth atrelatively high doses, e.g., higher than 5 mM, (so that its conversionto inosine is inhibited). At such concentrations, however, adenosine maybecome toxic. Adenosine analogs, e.g., 6-thioguanine are, therefore,preferable for administration in mammalian subjects to inhibit axonalgrowth. It is to be noted that dosage values may vary with the severityof the condition to be alleviated. It is to be further understood thatfor any particular subject, specific dosage regimens should be adjustedover time according to the individual need and the professional judgmentof the person administering or supervising the administration of thepurine nucleoside or analogue thereof and that dosage ranges set forthherein are exemplary only and are not intended to limit the scope orpractice of the claimed invention.

The invention, in another embodiment, provides a pharmaceuticalcomposition consisting essentially of a purine nucleoside or analogthereof and a pharmaceutically acceptable carrier and methods of usethereof to modulate axonal outgrowth by contacting CNS neurons with thecomposition. By the term “consisting essentially of” is meant that thepharmaceutical composition does not contain any other modulators ofneuronal growth such as, for example, nerve growth factor (NGF). In oneembodiment, the pharmaceutical composition of the invention can beprovided as a packaged formulation. The packaged formulation may includea pharmaceutical composition of the invention in a container and printedinstructions for administration of the composition for treating asubject having a disorder associated with an injury of central nervoussystem neurons, e.g., an injury to retinal ganglion cells, a spinal cordinjury or a traumatic brain injury.

In Vitro Treatment of CNS Neurons

CNS neurons can further be contacted with a therapeutically effectiveamount of a purine nucleoside or analog thereof, in vitro. Accordingly,CNS neuron cells can be isolated from a subject and grown in vitro,using techniques well known in the art. Briefly, a CNS neuron cellculture can be obtained by allowing neuron cells to migrate out offragments of neural tissue adhering to a suitable substrate (e.g., aculture dish) or by disaggregating the tissue, e.g., mechanically orenzymatically, to produce a suspension of CNS neuron cells. For example,the enzymes trypsin, collagenase, elastase, hyaluronidase, DNase,pronase, dispase, or various combinations thereof can be used. Trypsinand pronase give the most complete disaggregation but may damage theells. Collagenase and dispase give a less complete disaggregation butare less harmful. Methods for isolating tissue (e.g., neural tissue) andthe disaggregation of tissue to obtain cells (e.g., CNS neuron cells)are described in Freshney R. I., Culture of Animal Cells, A Manual ofBasic Technique, Third Edition, 1994, the contents of which areincorporated herein by reference.

Such cells can be subsequently contacted with a purine nucleoside oranalog thereof at levels and for a duration of lime as described above.Once modulation of axonal outgrowth has been achieved in the CNS neuroncells, these cells can be re-administered to the subject, e.g., byimplantation.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting. The contents of allreferences, patents and published patent applications cited throughoutthis application are hereby incorporated by reference.

EXAMPLES

In the following examples, the following methodologies were used:

Sample Preparation

Axogenesis factor-1 was obtained essentially as described in Schwalb etal., 1995, and Schwalb et al., Neuroscience, 72: 901-910, 1996, thecontents of which are incorporated herein by reference). Optic nerveswere dissected, cut into fragments 1 mm in length, and incubated in aratio of 6 nerves in 3 ml of either L-15 media (Gibco BRL) orphosphate-buffered saline (Gibco BRL). After 3-4 hours, nerve fragmentswere removed by filtering through a 0.22 μm pore low protein-bindingfilter (Gelman). A low molecular weight fraction of the conditionedmedia was prepared by ultrafiltration, first with a molecular weightcut-off of 3 kDa (Amicon Centriprep-3), then with a cut-off of 1 kDa(Filtron). The filtrate was used as a positive control at 20-30% finalconcentration. Adenosine, adenosine 5′ monophosphate, adenosinedeaminase, adenosine diphosphate, adenosine triphosphate, 8-bromo3′,5′-cyclic guanosine monophosphate, 3′,5′ cyclic adenosinemonophosphate, 5′ cyclic guanosine monophosphate, cytidine, guanosine,hypoxanthine, inosine, 5′-inosine monophosphate, a-tocopherol,6-thioguanine, thymidine, uridine, and xanthine were all obtained fromSigma Chemical Co., St. Louis, Mo., 8-p-sulphophenyl-theophylline,dibutyryl cyclic adenosine monophosphate and 2-deoxycoformycin were fromCalbiochem, 2-chloroadenosine, erythro-9-(2-hydroxy-3-nonyl) adenine andIB-MECA from Research Biochemicals, Inc. (Natick, Mass.), and4-(nitrobenzyl-6-thioinosine) from Aldrich Chemicals, Inc. Themembrane-permeable, nonhydrolyzable analogs of cAMP and cGMP,8bromoadenosine-3′,5′ cyclic monophosphorothioate and8-(4-chlorophenylthio) guanosine-3′,5′-cyclic monophosphate were fromBiolog.

Dissociated Retinal Cultures

Goldfish (Comet Variety, Mt. Parnell Fisheries, Mt. Parnell Pa.), 6-10cm in length, were dark-adapted and their retinas dissected. Retinaswere incubated with papain (20 μg/ml), activated with cysteine (2.8 mM)for 30 minutes at room temperature, then dissociated by gentletrituration. Repeated cycles of trituration and sedimentation yieldedcultures nearly homogeneous in ganglion cells, which are readilyidentified by their oval shape, phase-bright appearance, size (diameter15 μm), and extension of only 1 or 2 neurites of uniform caliber; thesecriteria have been verified by retrograde labeling (see Schwartz &Agranoff, Brain Res. 206: 331-343, 1981 and Schwalb et al., J.Neuroscience 15: 5514-5625, 1995, the contents of which are incorporatedherein by reference). Low density cultures were achieved by plating c.5×10³ cells/well into poly L-lysine coated, 24-well culture dishes(Costar, Cambridge, Mass.). Cells were maintained at 21° C. in serumfree, defined media containing insulin, selenium, transferrin, bovineserum albumin, catalase, superoxide dismutase, hormones, and vitamins inEagle's L-15 media as described in Schwalb et al., 1995, the contents ofwhich are incorporated herein by reference). Dissociated cultures ofpurified rat retinal ganglion cells were prepared by immunopanning asdescribed in Barres et al., Neuron, 1: 791-803, 1988, the contents ofwhich are incorporated herein by reference). In brief, retinas frompostnatal day 8 Sparague-Dawley rats were dissociated using papainactivated with cysteine. Macrophages were removed by incubation with ananti-rat macrophage antibody (Accurate) followed by immunopanning withan anti-rabbit 1gG antibody. Ganglion cells were isolated byimmunopanning with an anti-Thy-1 antibody, then dislodged with trypsinfor use in low-density cultures. Rat retinal ganglion cells weremaintained at 37° C. in a CO₂ incubator using the same medium describedabove except for the presence of 30 mM bicarbonate.

Experimental Design

In a typical experiment, samples were plated in quadruplicate inrandomized positions of a 24-well plate and the code was concealed toensure that growth was evaluated in a blinded fashion. Each experimentcontained 4 wells of a negative control (media plus supplements only)and 4 wells of a positive control (a standardized AF-1 sample of knownactivity). Growth and survival were assessed after 6 days for allganglion cells in 25 consecutive fields of each well using phasecontrast microscopy at 400× magnification (c. 150 ganglion cells countedper well). Extension of a process 5 cell diameters in length was thecriterion for growth, since it clearly distinguishes stimulated cellsfrom negative controls (see Schwalb et al., 1995). After the completionof counting, the code was broken, the data tabulated, and means andstandard errors were calculated for the 4 replicate wells of each sampleusing Cricket Graph (CA Associates, Islandia, N.Y.). Data werenormalized by subtracting the growth in the negative controls (usually4-5%) and dividing by the net growth in the positive controls. In themost favorable experiments, more than 50% of retinal ganglion cells(RGCs) exposed to AF-1 extended axons 5 cell diameters in length after 6days. Group comparisons were based upon pairwise, 2-tailed Student'st-tests. Several independent experiments were performed for mostsamples, as noted in the figure legends. In some cases, cell viabilitywas assessed with the dye 5,6-carboxyfluorescein diacetate. Cellsurvival is reported as the number of viable RGCs per high-poweredfield.

Example I Purine Induced Stimulation of Axonal Outgrowth from GoldfishRetinal Ganglion Cells

The low molecular weight growth factor AF-1, secreted by optic nerveglia, induced dramatic outgrowth from goldfish retinal ganglion cells.Little outgrowth occurred in the control condition using defined mediaalone. These two limits were the basis for normalizing results for otherfactors. When nucleosides were tested at concentrations between 1-100μM, adenosine and guanosine stimulated almost as much outgrowth fromgoldfish retinal ganglion cells as AF-1. Pyrimidine bases had noactivity over this concentration range. A more complete dose-responsecurve for the purines shows that adenosine is the more active of thetwo, with an EC₅₀ of 10-15 μM (see FIG. 1B). At concentrations of 50-100μM, adenosine induced a maximal response equal to 60% the level inducedby AF-1, but at higher concentrations, outgrowth decreased. Guanosinehad a higher EC₅₀ than adenosine (25 μM, see FIG. 1B), and atconcentrations of 100 μM, it stimulated the same maximal level ofactivity as adenosine, with no obvious decrease in activity at higherconcentrations.

Example II Purine Nucleotides are Less Active than Nucleosides

Extracellularly, adenosine could be stimulating either P₁ receptors,which are optimally responsive to adenosine per se, or P₂ receptors,which respond maximally to ATP Or other nucleotides. AMP and ADP showeda marginally significant level of activity at 100 μM (p 0.05), as didATP at 10 μM (but not at 100 μM). Since the activity of the purinenucleotides is considerably lower than that of the purines themselves,it is unlikely that P2 receptors are involved. Plausibly, the purinescould function intracellularly as precursors for cyclic nucleotides thatmight serve as second messengers in axogenesis. The biological activityof membrane-permeable analogs of cAMP and cGMP was, therefore, examined.Neither dibutyryl cAMP (dBcAMP) nor 8-Br cGMP showed any activitybetween 1-100 μM (see FIG. 1D). More recently developed nonhydrolyzable,membrane-permeable analogs of cAMP (8-bromoadenosine-3′,5′ cyclicmonophosphorothioate: Sp-8-Br-cAMPS) and cGMP (8-(4-chlorophenylthio)guanosine-3′,5′-cyclic monophosphate: 8-pcpt-cGMP)) were also found tobe inactive when tested at concentrations up to 1 mM (see FIG. 1D).

Example III The Positive Effects of Adenosine are not Mediated throughExtracellular Adenosine Receptors

8-p-(sulfophenyl theophylline) (8-PST), described in Collis et al.,Brit. J. Pharmacol. 92:69-75, 1987, the contents of which areincorporated herein by reference, is an inhibitor of the two most commonadenosine receptors (A1 and A2). At 20 μM, a dosage that almostcompletely blocks receptor-mediated effects of adenosine in rats, 8-PSThad no effect on outgrowth stimulated by adenosine, guanosine, or AF-1(see FIG. 2). Further evidence that the positive effects of adenosineare not mediated through extracellular adenosine receptors comes fromstudies using the non-hydrolyzable analog 2-chloroadenosine (2CA), whichis an agonist at the A1, A2 and A3 receptors. At concentrations of 10and 100 μM, 2-CA caused a small but significant decrease in growth belowthe baseline in 3 out of 3 independent experiments (see FIG. 2).

Example IV Adenosine Must be Hydrolyzed to Inosine to Stimulate Growth

To investigate whether the activity of adenosine is due to the formationof an active metabolite, the activity of ADA was inhibited using eitherdeoxycoformycin (DCF) or erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA).In the presence of 10 μM DCF, 100 μM adenosine not only failed tostimulate growth, but caused it to decline below baseline levels (FIG.3, lanes e vs. d). Cell survival also decreased when adenosinehydrolysis was blocked. In the presence of 10 μM DCF, 10 μM adenosinecaused survival to decrease by 20% (not shown), and 100 μM adenosinecaused survival to decline by 57% (FIG. 3, bottom, lane e). The effectsof DCF on outgrowth and survival were specifically related to thepresence of nonhydrolyzed adenosine, since they did not occur when DCFwas used alone, with AF-1, or with guanosine (FIG. 3, lanes b and h).Like DCF, 10 μM EHNA rendered adenosine (100 μM) ineffective instimulating outgrowth and caused cell survival to decline by about 30%(data not shown). EHNA also exhibited nonspecific effects, however,reducing growth stimulated by either guanosine or AF-1 by about 50%,though not altering cell survival. Further evidence that the positiveeffects of adenosine require its hydrolysis comes from experiments inwhich exogenous ADA was added. At 0.4 U/ml, the enzyme did not diminishaxon outgrowth stimulated by 100 μM adenosine or affect cell survival(see FIG. 3, lane f).

Example V Inosine is the Active Metabolite

Inosine, the primary product of adenosine deamidation, proved to be apotent activator of axon outgrowth. As shown in FIG. 4, the EC₅₀ forinosine was 10-15 μM, and a maximal response, equal to about 60% thelevel achieved with AF-1, was attained at concentrations above 25 μM.While the EC₅₀ and maximum response induced by inosine were similar tothose of adenosine, one notable difference was that at higherconcentrations, inosine did not cause growth to decline, unlike the casefor adenosine. Further hydrolysis of inosine yields hypoxanthine, whichshowed no activity at all (see FIG. 4). Inosine 5′ monophosphate (5′IMP) was inactive at 10 μM, and at 100 μM it showed less activity thaninosine at 10 μM (see FIG. 4).

Example VI Purines Stimulate Growth through an Intracellular Pathway

Two inhibitors of the purine transporter, nitrobenzylthio inosine (NBTI)and dipyridamole, were used to investigate whether inosine and guanosineneeded to enter neurons to stimulate outgrowth. At 20 μM, NBTI blockedabout 90% of the growth induced by either inosine or guanosine (see FIG.5; 86% loss of activity for 50 μM inosine, p<0.001; 93% loss of activityfor 100 μM guanosine, p<0.01). Dipyridamole (10 μM) also diminished thegrowth induced by inosine (114% decrease; p<0.01; not shown; guanosinenot tested). In contrast, AF-1 showed little inhibition by NBTI (10%decline, n.s.) and slightly more with dipyridamole (25% decline, n.s.,not shown). The NBTI-related loss in activity for the purines was fargreater than for AF-1 (p<0.001).

Example VII AF-1 Activity is not Due to Inosine

To address whether AF-1 preparations might still contain purines thatcould account for some of their biological activity, native AF-1 andinosine were chromatographed on a size-exclusion column with SephadexG-10 (Pharmacia Biotech, Uppsala, Sweden), 1 cm in diameter and 10 cm inlength. Samples were loaded in a volume of 0.5 ml and collected in 1 mlfractions. The column buffer was either 20% methanol in distilled wateror 0.14 M NaCl. Fractions were bioassayed at 30% concentration. As shownin FIG. 6A, the peak of inosine activity was at 9-10 minutes, whereasfor AF-1 it occurred at 7 minutes.

Example VIII Axonal Outgrowth is the Effect of Inosine and Guanosine andnot the Effect of a Secondary Factor

The cultures used here contained 70-90% ganglion cells, with theremainder representing other neural and non-neuronal elements of theretina (see Schwartz & Agranoff, 1982 and Schwalb et al, 1995, thecontents of which are incorporated herein by reference). Thisheterogeneity raised the possibility that inosine or guanosine could actfirst upon another cell population, which secretes a secondary factorthat stimulates retinal ganglion cells to grow. In this case, the effectof the purines would be expected to vary with cell density, since theconcentration of any secondary factor would increase proportionatelywith increasing density. To examine this, axonal outgrowth wasinvestigated in response to a fixed concentration of inosine orguanosine over a 3-4-fold range of cell densities. The regression linesfor both the inosine and the guanosine data demonstrate that growth isnot a function of cell density (see FIG. 6B), arguing against thepresence of a concentration-dependent secondary factor.

Example IX Induction of Phosphoprotein GAP-43 Expression by Purines

One hallmark of optic nerve regeneration in vivo is the enhancedexpression of the membrane phosphoprotein GAP-43. To investigate whetherthis upregulation is induced by purines, immunohistochemistry wascarried out using a polyclonal rabbit antiserum against recombinantgoldfish GAP-43. Recombinant zebrafish GAP-43 was made by transformingE. coli with a cDNA isolated by Dr. Eva Reinhard, University of Basel,Switzerland (see Reinhard et al., Development, 120: 1757-1775, 1994, thecontents of which are incorporated herein by reference) subcloned intothe prokaryotic expression vector pTrcHisB (Invitrogen). The proteinproduced was purified by Ni²⁺-NTA-affinity chromatography and used toimmunize rabbits. The specificity of the resulting antibody wasdemonstrated in western blots, where the antibody recognized a unique 48kDa band that is enriched in retinal ganglion cells undergoingregeneration or in synaptosomal plasma membranes from goldfish brain.

AF-1, inosine, and guanosine all caused a large increase in GAP-43levels relative to L-15 treated controls. A semi-quantitative analysiswas carried out by assigning a level for GAP-43 immunoreactivity of 0(none), 1 (moderate) or 2 (intense), and correlating the stainingintensity with the length of a cell's axon for 150-200 cells treatedwith L-15, inosine, or AF-1. Inosine produced a 5.5-fold increase in thenumber of intensely stained cells over L-15, whereas AF-1 produced a8-fold increase. In all 3 cases, the intensity of GAP-43 immunostainingcorrelated strongly with axonal length.

Example X Blockade of Axonal Outgrowth with 6-thioguanine (6-TG)

In goldfish RGCs, 6-TG at 10 μM blocked all growth stimulated by AF-1(see FIG. 7A, lane 2), but had no effect on cell survival (see FIG. 7B).The same concentration of 6-TG reduced outgrowth stimulated by 25 μMinosine by only 50% (see FIG. 7A, lanes 3 and 4), and had no effect ongrowth stimulated by either 100 μM inosine or 100 μM guanosine (see FIG.7A, lanes 5-8). At 100 μM, inosine fully restored the growth induced byAF-1 in the presence of 10 μM 6-TG back to its original level, which wassignificantly higher than the level of growth induced by inosine alone(see FIG. 7A, lanes 10 vs. 6). Therefore, inosine and 6-TG appear to beacting competitively at a level of intracellular signaling that is alsoutilized by AF-1 to stimulate outgrowth. Further evidence that inosinemay activate the same pathway that is utilized by AF-1 signaling camefrom the observation that when the two were combined at their EC₅₀levels, they showed additive effects, whereas at saturatingconcentrations growth saturates at the level stimulated by high AF-1levels alone (see FIG. 7C, lane 9). Since 6-TG has a free thiol, itcould be acting as a reducing agent rather than as a purine analog.However, two other reducing agents, a-tocopherol at 30 μM or glutathionea-methyl ester (MEG) at 100 μM had no effect on outgrowth stimulated byAF-1 (see FIG. 7D). Another possibility is that inosine might block theinhibitory effect of 6-TG on outgrowth by interfering with its transportinto cells. However, the two transport inhibitors that blocked theactivity of inosine, NBTI and dipyridamole, failed to prevent 6-TG fromblocking outgrowth stimulated by AF-1 (see FIG. 7D).

Example XI Mammalian Retinal Ganglion Cells Extend Axons in Response toInosine

Retinal ganglion cells were isolated from 8 day old rats byimmunopanning as described in Barres et al., Neuron, 1: 791-803, 1988,the contents of which are incorporated herein by reference, and grown indefined media. Inosine at 25 or 50 μM stimulated a 50% increase in thenumber of cells extending axons 5 cell diameters in length (see FIG. 8).Ciliary neurotrophic factor (CNTF) induced a much larger increase inoutgrowth (see FIG. 8) and enhanced cell survival. At 10 μM, 6-TGblocked CNTF-induced outgrowth. The addition of inosine at 50 μMrestored CNTF-induced outgrowth nearly to its original level (see FIG.8).

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method for stimulating axonal outgrowth of central nervous systemneurons in a mammal with a central nervous system injury, comprisinginjection of the mammal with a pharmaceutical composition consistingessentially of an effective amount of inosine to thereby contact theinjured central nervous system neurons with the inosine, such thataxonal outgrowth is stimulated.
 2. A method for stimulating axonaloutgrowth of central nervous system neurons in a mammal with a centralnervous system injury, comprising, injection of the mammal with apharmaceutical composition that consists of inosine and apharmaceutically acceptable carrier, to thereby contact the injuredcentral nervous system neurons with the inosine, such that axonaloutgrowth is stimulated.
 3. A method of treating a mammal havingsuffered a central nervous system injury, comprising injecting themammal with a pharmaceutical composition consisting essentially of aneffective amount of inosine to thereby contact the injured centralnervous system neurons with the inosine, such that axonal outgrowth isstimulated.
 4. A method of treating a mammal having suffered a centralnervous system injury, comprising injecting the mammal with apharmaceutical composition consisting of an effective amount of inosineto thereby contact the injured central nervous system neurons with theinosine, such that axonal outgrowth is stimulated.
 5. The method ofclaim 1 or 2, wherein the pharmaceutical composition contacts theinjured CNS neurons in vivo with a concentration of inosine of 5 uM to 1mM.
 6. The method of claim 1 or 2, wherein the mammal is a human.
 7. Themethod of claim 1 2, 3, or 4, wherein the central nervous system injuryis spinal cord injury, traumatic brain injury, or stroke.
 8. The methodof claim 7 wherein the spinal cord injury is selected from the groupconsisting of monoplegia, diplegia, paraplegia, hemiplegia andquadriplegia.